Acetyl-coenzyme A (acetyl-CoA) carboxylase (ACCase) is a biotinylated enzyme that catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate. Malonyl-CoA is an essential substrate for fatty acid biosynthesis in plastids (Harwood, J. L., Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:101-138 (1988)) and chain elongation in the cytosol (Pollard, M. et al., Plant Physiol. 66:649-655 (1980)). In addition, malonyl-CoA is required in the cytosol for several reactions including the biosynthesis of flavonoids (Ebel, J. et al., Eur. J. Biochem. 75:201-209 (1977) and Ebel, J. et al., Arch. Biochem. Biophys. 232:240-248 (1984)). It has been shown that the regulation of activity of the plastid ACCase is a major determinant of the flux of carbon into fatty acid synthesis in spinach (Post-Beittenmiller, D. et al., J. Biol. Chem. 266:1858-1865 (1991) and Post-Beittenmiller, D. et al., Plant Physiol. 100:923-930 (1992)) and in barley or maize leaves (Page, R.A. et al., Biochem. Biophys. 1210:369-372 (1994)). Two forms of ACCase, termed "prokaryotic" and "eukaryotic," have been isolated and characterized. The prokaryotic form is a heteromeric chloroplast enzyme (also referred to as a multi-subunit (MS) enzyme) composed of dissociable subunits. For example, E. coli ACCase is composed of four subunits: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP), and the carboxyltransferase (CT) .alpha.- and .beta.-subunits (Li, S-J. et al., J. Bio. Chem. 267:855-863 (1992); Li, S-J. et al., J. Bio. Chem. 267:16841-16847 (1992); Kondo, H. et al., PNAS (USA) 88:9730-9733 (1989) and Alix, J-H. et al., DNA 8:779-789 (1989)). The eukaryotic form of ACCase is a homomeric, presumably cytosolic enzyme (also referred to as multi-functional (MF) enzyme) containing the BC, BCCP, and CT functional domains with a molecular weight of more than 200 kDa. The full-length homomeric ACCase has been cloned from mammals (Takai, T. et al., J. Biol Chem. 263:2651-2657 (1988); Lopez-Casillas, F. et al., PNAS (USA) 85:5784-5788 (1988); Ha, J. et al., Eur. J. Biochem. 219:297-306 (1994)), yeast (Al-Feel, W. et al., PNAS (USA) 89:4534-4538 (1992)), algae (Roessler, P. G. et al., J. Biol. Chem. 268:19254-19259 (1993)) and plants (Shorrosh, B. S. etal., PNAS (USA) 91:4323-4327 (1994); Roesler, K. R. et al., Plant Physiol. 105:611-617 (1994) and Gornicki, P. et al., PNAS (USA) 91:6860-6864 (1994)). Partial homomeric ACCase sequences have also been reported from several plants (Elborough, K. M. et al., Plant Mol. Biol. 24:21-34 (1994) and Ashton, A. R. et al., Plant Mol. Biol. 24:35-49 (1994)). To date, all homomeric ACCase sequences reported from plants show high sequence identity in their encoded amino acid sequences. Thus, there is currently no sequence evidence for differences, if any, between plastidial and cytosolic forms of homomeric ACCase. Preliminary evidence suggested that the alfalfa and Arabidopsis homomeric ACCases are localized in the cytosol (Shorrosh, B. S. et al., PNAS (USA) 91:4323-4327 (1994) and Roesler, K. R. et al., Plant Physiol. 105:611-617 (1994)). However, two isoforms of the homomeric ACCase have been characterized from maize based on their chloroplastic and extra-chloroplastic localization and their herbicide sensitivity (Egli, M. A. et al., Plant Physiol. 101:499-506 (1993)).
Recently it has been shown that homomeric ACCase isolated from young pea leaves is localized in the epidermal tissues (Alban, C. et al., Biochem. J. 300:557-565 (1994)). In addition, Western blot analysis using biotin antibodies detected homomeric ACCase in the total protein extract from both Gramineae and dicot plants, but the homomeric ACCase was only present in the chloroplast of Gramineae plants thus reported (Konishi, T. et al., PNAS (USA) 91:3598-3601 (1994); Gornicki, P. et al., J. Bacteriology 175:5268-5272 (1993); Egli, M. A. et al., Plant Physiol. 101:499-506 (1993); Wurtele, E. S. et al., Plant Physiol. 99:1699-1703 (1992) and Baldet, P. et al., Arch. Biochem. Biophys. 303:67-73 (1993)). Recently, immunological data describing the expression of a pea chloroplast encoded protein, which shares sequence similarity with the E. coli carboxyltransferase .beta.-subunit (accD) of ACCase was reported (Sasaki, Y. et al., J. Biol. Chem. 268:25118-25123 (1993)). Antibodies against the pea accD-like protein precipitated the activity of pea chloroplast ACCase with the concomitant precipitation of three polypeptides including a 35-kDa biotin-containing protein (Sasaki, Y. et al., J. Biol. Chem. 268:25118-25123 (1993)). Also, a heteromeric ACCase enzyme consisting of dissociable subunits with molecular weights ranging from 32 to 79 kDa has been partially purified and characterized from the epidermal and mesophyll tissues of pea leaves. One of these subunits, with a molecular weight of 38 kDa, was biotinylated (Alban, C. et al., Biochem. J. 300:557-565 (1994)). Western blot analysis of pea chloroplasts localized only one biotin-containing protein with an apparent molecular weight of 35 to 38 kDa (Sasaki, Y. et al., J. Biol. Chem. 268:25118-25123 (1993) and Baldet, P. et al., Arch. Biochem. Biophys. 303:67-73 (1993)); others have reported multiple biotin-containing proteins in dicot chloroplasts (Gornicki, P. et al., J. Bacteriology 175:5268-5272 (1993) and Wurtele, E. S. et al., Plant Physiol. 99:1699-1703 (1992)). Taken together, the reported data suggest that dicot plastids contain heteromeric ACCase; the number of subunits and their organization, however, is not yet understood.
It would thus be desirable to provide the biotin carboxylase subunit of acetyl-CoA carboxylase. It would also be desirable to control expression of the gene encoding the biotin carboxylase subunit of acetyl-CoA carboxylase. It would further be desirable to control the carboxylation of acetyl-CoA to produce malonyl-CoA. It would also be desirable to control the carboxylation of acetyl-CoA to produce malonyl-CoA by controlling the expression of genes encoding the ACCase subunits. It would further be desirable to acquire long-term control of the carboxylation of acetyl-CoA to produce malonyl-CoA by genetically altering plants. It would also be desirable to control fatty acid synthesis in plants and seeds by controlling the carboxylation of acetyl-CoA to produce malonyl-CoA. It would further be desirable to control fatty acid synthesis in plants and seeds without employing foreign chemicals.